Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes. These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins. By forming a continuous protein pathway across the membrane, these proteins enable the specific molecules to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer of the plasma membrane.
There are two major classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins bind the specific molecule to be transported and undergo a series of conformational changes in order to transfer the bound molecule across the membrane. Channel proteins, on the other hand, need not bind the molecule. Instead, they form hydrophilic pores that extend across the lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Transport through channel proteins occurs at a much faster rate than transport mediated by carrier proteins.
Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the membrane are referred to as voltage-sensitive ion channels.
The sodium channel is one of the most thoroughly characterized of the voltage-sensitive channels (see FIG. 1 for a model of a voltage-sensitive sodium channel). In vertebrates, sodium channels in the brain, muscle, and other tissues are large membrane glycoprotein complexes composed of an alpha subunit (230-270 kDa) and 1-2 tightly associated smaller (33-38 kDa) beta subunits (reviewed by Catterall 1992). The large alpha subunit forms the ion permeable pore while the smaller subunits play key roles in the regulation of channel function (Isom et al. 1992; reviewed by Isom et al. 1994). The alpha subunit is common to purified channel preparations from Electrophorus electricus (electric eel) electric organ (Noda et al. 1984), rat brain (Noda et al. 1986), rat skeletal muscle (Barchi 1988) and chick heart muscle (Catterall 1986). Other studies have revealed the existence of multiple closely related isoforms of the sodium channel found in different animal species, in different tissues within the same species, and even in the same tissue (Catterall et al. 1981; Frelin et al. 1984; Rogart 1986; Moczydlowski et al. 1986).
The structure of invertebrate sodium channels is not as well defined. Gene cloning studies have established the existence of alpha subunits of structure similar to those described for vertebrates (Loughney et al. 1989; Ramaswami and Tanouye 1989; Okamoto et al. 1987). Analysis of the para behavioral mutant (paralytic; Suzuki et al. 1971) of Drosophila melanogaster revealed that the para gene encodes a Drosophila sodium channel alpha subunit (Loughney et al. 1989). The entire para cDNA sequence was determined (Loughney et al. 1989; Thackeray and Ganetzky 1994).
The kdr mutant of the house fly Musca domestica has also been studied. The kdr insecticide resistance trait of the house fly confers reduced neuronal sensitivity to the rapid paralytic and lethal actions of DDT and pyrethroid insecticides (Soderlund and Bloomquist 1990). Because these insecticides are known to modify neuronal excitability by altering the inactivation kinetics of voltage-sensitive sodium channels (Soderlund and Bloomquist 1989; Bloomquist 1993), efforts to identify the molecular basis of kdr resistance have focused on the pharmacology and structure of this target.
Recently, tight genetic linkage between the kdr trait and a restriction fragment length polymorphism located within a segment of the house fly homolog of the para gene of Drosophila melanogaster was demonstrated (Knipple et al. 1994). Similar linkage studies have also documented tight linkage of the super-kdr resistance trait of the house fly (Williamson et al. 1993) to molecular markers lying within the para-homologous voltage-sensitive sodium channel gene.
Elucidation of the structure of the house fly sodium channel gene will enable the screening of potential insecticidal agents which act upon the sodium channel.
A need continues to exist, therefore, for the determination of the primary structure of the house fly sodium channel, i.e. the nucleotide and amino acid sequences of the channel.